Creatine (C4H9O2N3 or α-methyl guanidine-acetic acid ) is a compound present in vertebrate muscle tissue, principally as phosphocreatine. Creatine is synthesized primarily in the liver and also in the pancreas and the kidneys. Creatine helps produce energy needed to contract muscles and it is produced at a relatively constant rate. Creatine is eventually spontaneously degraded into creatinine by muscle and is released into the blood. It is then excreted by the kidneys and removed by the body by glomerular filtration.
The amount of creatinine produced is relatively stable in a given person. Serum creatinine level is therefore determined by the rate it is being removed, which is roughly a measure of kidney function. If kidney function falls, serum creatinine level will rise. Thus, blood levels of creatinine are a good measure of renal function. Usually, increased creatinine levels do not appear unless significant renal impairment exists. Tietz N W, Fundamentals of Clinical Chemistry, 2nd Edition, W B Saunders Company, 1982, pg. 994–995.
According to the American Diabetes Association (ADA), 20%–30% of patients with diabetes develop diabetic kidney disease (nephropathy). Further, some authorities recommend measurement of serum creatinine levels in non-diabetic patients to screen for renal dysfunction because of increasing evidence that dietary protein restriction and use of angiotensin-converting enzyme (ACE) inhibitors can retard progression once renal insufficiency develops. Thus, the need for creatinine testing as a measure of kidney function is well established.
Most methods for measuring creatinine levels are based upon the “Jaffe reaction” (1886), named after the German biochemist Max Jaffe (1841–1911), who discovered the reaction between creatinine and picrate ion in an alkaline medium that results in the formation of an orange-red complex. In 1904, Otto Folin adapted this method for use with urine. Folin, O. Physiol Chem. 1904, 41:223.
Several enzymatic methods are known for determination of creatinine in serum or urine, but they suffer from the disadvantage that they either proceed via creatine or ammonia as intermediate products in the reaction sequence. Since these substances are present in the serum or urine sample to be analyzed in concentrations which are quite substantial relative to creatinine, it is necessary to carry out differential measurements on two separate or successive reaction mixtures, the first of which the free creatine or ammonia is determined and the second of which the portion of additional creatine or ammonia formed from creatinine is determined. Such methods are relatively complicated for manual procedures and their application to automated analytical systems is also very limited, particularly when longer incubation periods are necessary for the completion of the conversion reactions.
U.S. Pat. No. 4,816,393 (Roche) discloses an enzymatic method for the determination of creatinine or 1-methylhydantoin. The substance 1-methylhydantoin is hydrolyzed to N-carbamoylsarcosine using the enzyme disclosed in the '393 patent, 1-methylhydantoinase (NMHase), which requires a nucleoside triphosphate, preferably ATP, as well as divalent metal ions and in some circumstances K+ ions and/or NH4 ions for its activity. Significantly, by using NMHase, a reaction pathway is provided, as shown below, that does not involve any intermediates found in appreciable concentrations in human blood.

Steps 3–5 of the above reaction pathway are disclosed in U.S. Pat. No. 4,645,739 (Roche), wherein a process for making the enzyme N-carbamoylsarcosine-amidohydrolase (CSHase) is disclosed. A corresponding photometric method for the determination of creatinine is also described in J. Siedel et al., Anal. Letters 21, 1009–1017 (1988). Endogenous substances present in body fluids should not interfere with this creatinine or 1-methylhydantoin determination since 1-methylhydantoin and the reaction products of the subsequent indicator reaction are not natural components of serum or urine. Therefore, a sample blank measurement should not be necessary.
Unfortunately, however, commercially available NMHase, which is needed to catalyze the reaction, requires enzyme-bound 1-methylhydantoin (NMH) for its stability. That is, NMHase is stabilized by its own substrate. According to U.S. Pat. No. 5,374,546 (Roche), in the presence of a nucleoside triphosphate and divalent metal ions, such as Mg2+, the NMH is completely degraded enzymatically and causes a blank reaction similar to an endogenous substrate. According to the '546 patent, “since the blank reaction caused by enzyme-bound NMH depends on the one hand on the amount of NMHase used and on the other hand also on the varying NMH content of the NMHase itself, it is also necessary to always determine the blank reaction separately for such a test and this blank value has to be subtracted from the measured value.” '546 patent, column 2, lines 65–68 (emphasis added). The '546 patent discloses a method for stabilizing NMHase after the NMH substrate has been removed. According to the '546 patent, NMH can be removed, the stabilization procedure for NMHase employed, and creatinine concentration can be tested using the reaction pathway shown above, without requiring a blank reaction.
With reference to FIG. 1, the prior art creatinine measuring device of the '546 patent includes supporting layer 1, layers 2 and 3 which are impregnated with components of the reagent system, a transport layer 4 preferably made of glass fibers, and blood separation layer 5, also preferably made of glass fibers. In use, 30 μl of blood is applied to layers 6 and 5. The sample seeps into transport layer 4 where red blood cells are separated during tangential (right to left) fluid migration. A reaction takes place in layers 2 and 3 between the constituents of the sample which is to be determined and the reagents impregnated in the layers 2 and 3. At a predetermined time after applying the sample, the indicator reaction is determined photometrically.
The stabilized NMHase is impregnated in layer 2 along with ATP, MgCl2, and a complexing agent (EDTA) that complexes the metal ions which are necessary to stabilize the NMHase. The indicator, a “Julolidino” indicator whose preparation is described in U.S. Pat. No. 4,665,023, is impregnated into layer 3, along with additional MgCl2 and NH4Cl.
Unfortunately, the inventors of the present invention have found two major drawbacks with the stabilization procedure disclosed in the '546 patent. First, they found it difficult to accomplish. Second, the “Julolidino” indicator disclosed in the patent is not commercially available. The '023 patent suggests that creatinine and uric acid assays are especially prone to interferences and require improved indicators, such as the Julolidino indicators. Further, a Reflotron Uric Acid bulletin applicants have reviewed states “Boehringer Mannheim has improved upon this procedure by including a special indicator, allowing evaluation of the reaction by reflectance photometry.” At any rate, the applicants of the present invention were influenced by the literature just mentioned, and feared that efforts to create a creatinine test assay would fail without using the Julolidino indicators and stabilization procedures taught by the prior art.
It would be desirable to have a creatinine test assay using NMHase that avoids the elaborate stabilization procedure and that uses conventional, off-the-shelf chromogens.